In light of the many recent theories regarding the involvement of reactive oxygen species in various disease states (ALS, arthritis, ischemia/reperfusion damage, cancer), elucidation of the mechanisms by which cells manage the catalytic metals known to produce oxidative damage has become a critical issue. The research described in the proposed project examines the potential copper-delivery function of two cytosolic proteins, HAH1 and CCS1. The hypothesis is that these two proteins transport copper ions from the membrane transport protein to the antioxidant enzyme Cu,Zn superoxide dismutase (SOD). Expression of the proteins in E. coli cells will provide sufficient quantities for structural characterization as well as evaluation of metal binding affinities and metal transfer chemistry. Mercury-199 NMR and X-ray absorption will be used to probe the structure of the metal binding site. Facilitation of copper insertion into apo-SOD by these proteins will be evaluated by assays (cytochrome c, NBT) of SOD activity during the copper-transfer event. Other proteins acting as complements in metal transfer will be sought if HAH1 and CCS1 do not directly facilitate copper insertion.